The purpose of this project is to develop and characterize a transgenic mouse model of familial focal segmental glomerulosclerosis (FSGS) using a podocyte-targeted transgene encoding a mutant actinin-4 linked to the disease in humans. Glomerulosclerosis is caused by many agents, including viral, toxic, pharmacologic, and hereditary mechanisms. With respect to the latter, most of the known mutations linked to human familial glomerulosclerosis cause defects in proteins specific to, or of unique importance in the glomerular podocyte. Three different point mutations in actinin-4 have been conclusively linked to an autosomal dominant form of FSGS. Actinin-4 is a protein which is expressed at high levels in most nonmuscle cells. The FSGS-causing mutations in actinin-4 have been shown to cause enhanced binding of actinin-4 to actin. Since actinin-4 and actin are widely co-expressed in a number of tissues, and there is no defect other than FSGS that occurs in persons inheriting mutant actinin-4, it seems unlikely that the enhanced binding of actinin-4 to actin is the cause of FSGS in the families affected by the mutations. Otherwise, cell types other than podocytes would be affected. The underlying goal of this project therefore is to determine the podocyte-specific mechanisms by which the mutations in actinin-4 cause FSGS. Toward this end, six lines of transgenic actinin-4 mutant mice have been produced, and two will be selected for this study based on high renal expression of thetransgene. The present proposal is: 1) to characterize a mouse model of glomerulosclerosis using transgenic mice expressing mutant actinin-4 in their podocytes, and 2) to perform complementary studies in mouse podocytes retrovirally transduced with an actinin-4/GFP fusion protein incorporating either mutant or wild type actinin-4. Studies to characterize thetransgenic mice will include monitoring urinary protein excretion over the normal lifespan of the mice, micro-array analysis of gene expression, histology of the kidney, and renal stress-inducing manipulations. Complementary studies in cultured podocytes expressing fluorescently matched expression levels of mutant or wild type actinin-4 will include fluorescent and confocal microscopy, micro-array gene expression studies and yeast two-hybrid analysis of podocyte cDNA using mutant and wild-type actinin-4 as bait.